Infectious disease represents a perennial threat to the general health and welfare of humankind. Unfortunately, the discovery of new antimicrobial drug leads has slowed in recent years due in part to a lack of novel chemical entities capable of effectively combating infectious organisms. Consequently, there is a critical need for developing new sources of compounds that can be tested to identifying novel bioactive pharmacophores. In this project, we propose to investigate the drug-lead discovery potential of a vast, but under-explored reservoir of secondary metabolites: bacteria from mammals. Similar to humans, the microbiomes of other mammals are composed of thousands of bacterial species and many of these organisms have the capacity to generate secondary metabolites. Despite this incredible potential, virtually no research effort has been reported regarding which microbiome-derived bacteria are capable of producing unique secondary metabolites, nor have their applications to the bioactive lead generation process been tested. We propose an exploratory R21 research program that combines the comprehensive cultivation of mammalian microbiomes with chemical and molecular methodological approaches to critically evaluate these microbial assemblages for the discovery of new antimicrobial natural products. Our proposed program is founded on a synergistic collaboration between a natural products chemistry group and microbial ecology laboratory. This will enable us to test the central hypothesis that the microbiomes of mammals contain an untapped wealth of secondary-metabolite-producing bacteria and determine the capacity for these compounds to inhibit the growth of bacterial and fungal pathogens. We will test a range of cultivation techniques to produce unique mammalian microbiome libraries that will be systematically screened to identify bioactive hits. A subset of hits will be selected based on a combination of chemical, taxonomic, and genomic datasets for scale-up testing and bioactive secondary metabolite purification/characterization. This research is significant because it will provide a new resource for securing natural-product-based bioactive leads, which we will use to help invigorate the antimicrobial drug discovery pipeline. We expect that mammalian microbiomes will become a valuable resource for identifying novel organic substances that are key to the success of modern drug development programs.